Purpose:
Toll-like receptor (TLR)-mediated innate immune responses mounted against invading pathogens, if left unchecked, can lead to undesirable inflammation and tissue damage. There is growing interest in identifying mechanisms or factors that modulate these responses. This study was undertaken to test the hypothesis that the membrane-associated mucins (MAMs) MUC1 and MUC16, expressed on the apical surface of the corneal epithelium, counter-regulate innate immune responses by limiting pathogen sensing and signaling by TLRs.

Methods:
In in vitro experiments, a telomerase-transformed, immortalized human corneal-limbal epithelial (HCLE) cell line, grown to confluence and allowed to stratify for optimal mucin expression, was used. Additionally, HCLE cells in which the MAMs MUC1 and MUC16 were independently and stably knocked down were used. Non-transfected and knockdown HCLE cells were exposed to agonists specific to TLR2 (heat-killed Listeria monocytogenes) and TLR5 (flagellin), since these receptors were found to be the most abundantly expressed by stratified HCLE and native corneal epithelial cells. As an outcome of TLR activation and signaling, expression of the pro-inflammatory cytokines (IL-6 and TNF-α) and chemokine (IL-8) was assayed by performing qRT-PCR and immunoblotting. To corroborate in vitro findings, ex vivo experiments were performed wherein eyes derived from Muc1-/- null and Muc1-/-Muc4-/-Muc16-/- null mice were exposed to the TLR2 and TLR5 agonists and transcript levels of IL-6 and TNF-α were quantified in debrided corneal epithelia.

Results:
Following exposure to the TLR agonists: 1) MUC1 and MUC16 knockdown HCLE cells, in comparison to control HCLE cells, exhibited increased message levels of IL-6, IL-8, and TNF-α; knockdown cells were also found to secrete higher levels of IL-8 into the culture medium. 2) IL-6 and TNF-α mRNA expression was increased in the corneal epithelia of Muc1-/- null and Muc1-/-Muc4-/-Muc16-/- null mice in comparison to WT mice. In the absence of stimulation by TLR agonists, expression of IL-6, IL-8, and TNF-α among all the HCLE cell lines and mouse corneal epithelia remained comparable.

Conclusions:
Our results suggest that the MAMs MUC1 and MUC16 contribute to the maintenance of immune homeostasis at the ocular surface by suppressing TLR-mediated innate immune responses.